Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that modulate excitability in several brain regions involved in the pathogenesis of epilepsy, including hippocampus, neocortex, and thalamus. The preponderance of evidence shows that downregulation of Ih, the current generated by HCN channels, is associated with neuronal hyperexcitability and epilepsy. In the prior funding period of this project, the onset of epilepsy in an animal model was found to be associated with loss of HCN channel expression, and downregulation of HCN channel gating (i.e. hyperpolarization of Ih voltage-dependent activation). This latter change in HCN channel properties may be particularly important in the generation of seizures, as lamotrigine, an antiepileptic drug, produces upregulation of HCN channel gating as part of its antiepileptic action. A novel modulator of HCN channel gating, p38 mitogen- activated kinase (p38 MAPK), was characterized as well, with inhibition of p38 MAPK causing downregulation of HCN gating. In the present proposal, the links between phosphorylation (i.e. kinase or phosphatase) signaling and HCN channels will be explored. The overall hypothesis is that the downregulation of HCN channel gating that occurs in epilepsy may be due to loss of kinase activity, such as that of p38 MAPK, or to increased phosphatase activity. We will also explore whether direct modulation of p38 MAPK activity exerts an antiepileptic action in vivo. Specifically, we will answer the following questions: 1) Is altered HCN channel gating in epilepsy associated with loss of kinase activity? 2) Is altered HCN channel gating in epilepsy associated with increased phosphatase activity? 3) Is upregulation of HCN gating via activation of p38 MAPK signaling effective in an animal model of temporal lobe epilepsy (TLE)? To answer these questions, we will use cellular electrophysiology techniques in the brain slice preparation, biochemical techniques to assay kinase and phosphatase activity, and long-term video-electroencephalography (VEEG) in the pilocarpine animal model of epilepsy. The outcome of these experiments should increase our understanding of the molecular mechanisms by which seizures alter ion channel biophysical properties, determine whether epilepsy is associated with derangement of phosphorylation signaling pathways, and explore possible novel antiepileptic treatment strategies. PUBLIC HEALTH RELEVANCE: The current proposal will study the mechanisms of epilepsy at cellular and molecular levels. Epilepsy is one of the most common neurological diseases, affecting nearly 1% of the population, and causing significant disability in the 30% of epilepsy patients whose seizures are uncontrolled by existing medication. The outcome of this study may identify specific biochemical pathways that could be targeted for development of novel antiepileptic drugs, improving the likelihood that currently poorly-controlled patients may one day be seizure-free.